ARTICLES
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Ancient T-independence of mucosal IgX/A:
gut microbiota unaffected by larval
thymectomy in Xenopus laevis
S Mashoof1, A Goodroe1, CC Du1, JO Eubanks1, N Jacobs1, JM Steiner2, I Tizard3, JS Suchodolski2 and
MF Criscitiello1
Many studies address the influence of the gut microbiome on the immune system, but few dissect the effect of T cells on
gut microbiota and mucosal responses. We have employed larval thymectomy in Xenopus to study the gut microbiota
with and without the influence of T lymphocytes. Pyrosequencing of 16S ribosomal RNA genes was used to assess
the relative abundance of bacterial groups present in the stomach, small and large intestine. Clostridiaceae was the
most abundant family throughout the gut, while Bacteroidaceae, Enterobacteriaceae, and Flavobacteriaceae also
were well represented. Unifrac analysis revealed no differences in microbiota distribution between thymectomized and
unoperated frogs. This is consistent with immunization data showing that levels of the mucosal immunoglobulin IgX are
not altered significantly by thymectomy. This study in Xenopus represents the oldest organisms that exhibit class switch
to a mucosal isotype and is relevant to mammalian immunology, as IgA appears to have evolved from IgX based upon
phylogeny, genomic synteny, and function.
INTRODUCTION
The thymus is the primary T-lymphoid organ of vertebrates
from sharks to mammals.1,2 In humans, a small deletion on
chromosome 22 in DiGeorge syndrome often results in an
absent or hypoplastic thymus, with resulting loss of T-mediated responses (reviewed in ref. 3). The hairless “nude” strain
of mouse has an absent or greatly degenerated thymus owing
to a mutation in the Foxn1 gene.4 These mice do have B cells
but T cells are very few. Owing to the lack of both cytotoxic
and helper T cells, nude or thymectomized mice have abolished
allograft and mixed leukocyte reactions, proliferative responses
to classical T-cell mitogens, and antibody responses against
T-dependent antigens.5
The mucosal immune system forms the largest vertebrate
immune compartment and is mediated by specialized cells and
immunoglobulins, such as plasma cells producing secretory IgA
in birds and mammals. IgA production in the gut is not constitutive, shown by the absence of both IgA and IgA-secreting
plasma cells in the lamina propria of germ-free mice.6 There
are many studies investigating the humoral mucosal immune
responses of mammals lacking T cells, but they have yielded
mixed results.7,8 However, a picture is emerging of a significant T-cell-independent mechanism of gut IgA management
of mutualistic flora.9,10 Gut IgA-producing plasma cells in
mammals employ tumor necrosis factor- and inducible nitric
oxide synthase usually associated with innate phagocytes.11
Interestingly, B cells of lower vertebrates have been found to
have strong phagocytic activity.12 The B-cell phagocytic activity is consistent with an emerging theme of primitive, innate,
T-independent, IgA-switched B cells in the gut, although this
has never been actually tested in a lower tetrapod. This gap in
our knowledge prompted our assessment of the T-dependence
of humoral mucosal immunology in a phylogenetically relevant
model species.
The African clawed frog Xenopus laevis belongs to the
tongue-less frog family Pipidae. It is a choice model for
ontogeny and phylogeny of both humoral and cell-mediated
immunity. Xenopus shares a common ancestor with
1Comparative Immunogenetics Laboratory, Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University,
College Station, Texas, USA. 2Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas
A&M University, College Station, Texas, USA. 3Schubot Exotic Bird Health Center, Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical
Sciences, Texas A&M University, College Station, Texas, USA. Correspondence: MF Criscitiello ([email protected])
Received 13 April 2012; accepted 6 July 2012; published online 29 August 2012. doi:10.1038/mi.2012.78
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Normal
blood
β2M
TCRδ
TCRβ
TCRα
β2M
TCRδ
TCRβ
TCRα
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Tadpole gut
Normal gut
THX
blood
THX gut
Figure 1 Larval thymectomy greatly depletes TCR and message from the adult frogs. (a) Unilateral thymectomy at day 9 shows absence of
naturally melanized thymus on the left compared with intact organ on the right. Experimental frogs were bilaterally thymectomized. Image captured
at day 20 at original magnification ×3. (b) PCR contrasting levels of TCR, , and to 2-microglobulin in peripheral blood of three intact postmetamorphic frogs and three thymectomized post-metamorphic frogs. Results of 40 cycles of amplification. (c) Reverse transcriptase PCR comparing
the same amplicons from intestine of 9-day tadpoles (the age of thymectomy, gut of 10 animals pooled per row) and two intact post-metamorphic frogs
and two thymectomized post-metamorphic frogs.
mammals 350 million years ago and links them to the more ancient
vertebrates where the adaptive immune system arose (reviewed
in ref. 13). The ability to perform thymectomy on transparent
early stage Xenopus tadpoles made this frog an ideal model
species to query the thymic-dependent management of gut
microbiota and mucosal antibody responses from a fundamental
point in vertebrate humoral immunity (reviewed in ref. 14).
This present study is the first investigation determining
the gut bacterial populations of an amphibian using 454 pyrosequencing of the 16S ribosomal RNA (rRNA) gene. In addition, we examined the flora of normal and thymectomized
frogs, and the ability of thymectomized frogs to make mucosal
antibody responses. IgX has been functionally associated
with mucosal responses and (in contrast to IgY) was found in
thymectomized frogs.15,16 However, evolutionarily it has been
thought to be closer to IgM.17 T-independent responses are
known from Xenopus serum,18 but here we aimed to determine
the effect of thymectomy upon the gut flora, the mucosal and
the systemic IgX responses. We also evaluated the relationship
of amphibian IgX to mammalian IgA, in hopes of resolving
ambiguity as to the origins and natural history of the class of
antibody that manages vast numbers of mutualistic microbes
and is the first defense of the barriers breached by most
pathogens.
RESULTS
Larval thymectomy depletes T cells in the adult
To study the role of T cells in the management of gut bacterial
communities and mucosal humoral immunity from a fundamental standpoint in vertebrate evolution, we used the frog
larval thymectomy model. We performed thymectomies largely
executed as in the original studies,19 but 2 days later in development than in the original protocol owing to slightly slower
development of the larvae in our system. No frogs used in the
thymectomized group showed any thymic regrowth after cauterization (using microscopic inspection). Thymectomy greatly
diminished the detectable number of TCR transcripts using
constant region PCR in the adult frog. TCR and amplicons
could be detected in some frogs, but at a much lower frequency
MucosalImmunology | VOLUME 6 NUMBER 2 | MARCH 2013
than in frogs that had not undergone surgery (Figure 1b).
Regardless, larval bilateral thymectomy almost completely
ablated expression of TCR, showing that the classic T-cell
compartment has been removed from the experimental gut
model.
Pyrosequencing reveals diverse gut bacterial flora in
amphibian
DNA was prepared from frog luminal contents at three distinct locations in the gastrointestinal tract, which is relatively
simple in poikilothermic frogs and reptiles as they have lower
metabolic rates compared with most mammals and birds
(Supplementary Figure S1 online). Pyrosequencing of the
16S rRNA gene resulted in a total of 51,992 quality-sequencing
tags (mean, range: 2,888, 1,870–5,314). Figure 2 illustrates the
rarefaction curves for all samples at 1,800 sequences, suggesting sufficient coverage. Although rare operational taxonomic
units may have been identified with a higher sequencing depth,
the number of sequence tags used here is deemed adequate
to allow comparison of the beta diversity between samples.20
Samples taken from large intestine showed the richest diversity
of flora. Individual frogs showed distinct microbiota composition (Figure 3). Four frogs yielded similar rarefaction curves,
whereas one non-thymectomized frog (N321) showed more
diversity and one non-thymectomized (N571) less. Averaged
thymectomized samples gave a very similar rarefaction plot as
those from normal animals. These data allowed us to gain an
initial molecular description of the base amphibian gastrointestinal microbiota and look for differences between anatomic
sites and surgical groups.
Frog gut flora is anatomically distinct but not altered by
T cells
Many of the prokaryotic groups that dominate the human flora
are also major components in the frog flora. Clostridiaceae
dominated this amphibian community, and Bacteroidaceae
and Enterobacteriaceae were abundant in our sequencing
(Figure 3, Supplementary Table 3 online). In contrast to terrestrial mammalian flora, the environmental Flavobacteriaceae
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b
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Rarefaction measure: chao1
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Figure 2 Rarefaction analysis of 16S ribosomal RNA gene sequences obtained from frog gastrointestinal content. The analysis was performed
on a randomly selected subset of 1,800 sequences per sample. Lines represent the average of each sample type. (a) Stomach, orange; large intestine,
red; and small intestine, blue. (b) Normal (no surgery), blue and thymectomized, red. (c) Frog N011, red; N321, blue; T258, green; T606, yellow;
T339, brown; and N571, orange.
100%
Bacillaceae
Marinilabiaceae
90%
Flexibacteraceae
80%
Nostocaceae
Coriobacteriaceae
70%
Oscillatoriaceae
Lachnospiraceae
60%
Moraxellaceae
Enterobacteriaceae
50%
Rikenellaceae
40%
Fusobacteriaceae
Ruminococcaceae
30%
Erysipelotrichaceae
Porphyromonadaceae
20%
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10%
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Synergistaceae
0%
21 571 011 258 606 339 321 571 011 258 606 339 321 571 011 258 606 339
T
N T
T
T
T
T
T
T
N N
T
N
N
N N N
N3
Normal
Stomach
Thx
Normal
Thx
Small intestine
Normal
Thx
Desulfovibrionaceae
Flavobacteriaceae
Clostridiaceae
Large intestine
Figure 3 Bacterial families in the gastrointestinal tract of X. laevis. Familial distribution is shown with different colors from individual samples,
grouped by thymus status from each of the three sampled positions in the gastrointestinal tract. Families with at least 1% representation in any sample
are listed at the right. Complete taxonomic data is in Supplementary Table S2 online. Clostridiaceae were the predominant family, and no differences
in bacterial groups between normal and thymectomized frogs were observed.
constituted nearly a third (32.34%) of the stomach flora of one
frog and comprised 6.20 and 4.20% in two other individuals.
The Synergistaceae, Desulfovibrionaceae, Erysipelotrichaceae,
Ruminococcaceae, Rikenellaceae, and Porphyromonadaceae
also were substantial contributors (over 5% in at least one
sample) to the X. laevis microbiota.
The principal coordinates analysis plots based on the
unweighted UniFrac distance metric indicated that the
X. laevis stomach is composed of distinct microbial communities compared with the small and large intestine (Figure 4a).
This was most pronounced in the greater representation
of Flavobacteriaceae in the stomach compared with the more
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distal sites. Oscillatoriaceae cyanobacteria and Enterobacteriaceae
were in greater abundance in the stomach. This latter group
includes the common Gram-negative and sometimes pathogenic
Salmonella, Escherichia coli, Klebsiella, Shigella, and Yersinia
pestis more commonly associated with the mammalian intestine.
Synergistaceae was found in the intestines more than the
stomach, particularly in the small intestine of thymectomized
frog # 258. However, principal coordinates analysis plots
based on the unweighted UniFrac did not reveal differences
between small and large intestinal microbiota and, importantly, between normal and thymectomized frogs (Figure 4b).
The similar microbiological findings in the guts of normal and
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PCoA—PC1 vs. PC2
PCoA—PC1 vs. PC3
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a
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Figure 4 Principal coordinates analysis (PCoA) of unweighted UniFrac distances of 16S ribosomal RNA gene sequencing. The analysis was
performed on a randomly selected subset of 1,800 sequences per sample. (a) Stomach (blue circles) samples separated from small (green
square) and large (red triangle) intestine, indicative of distinct flora. (b) No such separation was seen between non-thymectomized (blue circle) and
thymectomized (red square) samples.
T-cell-depleted frogs prompted investigations of the mucosal
humoral immune compartment in this model.
of 1,000 bootstrap replications). This finding provided confidence
in assaying IgX in the frog as an ortholog as well as a functional
analog of mammalian IgA in mucosal immunity.
Amphibian IgX is likely orthologous to mammalian IgA
To assess the T-dependence of humoral immunity in the frog
alimentary canal, we wanted to be more confident of the mucosal
immunoglobulin in this amphibian. As more immunogenetic data
have recently become available from reptile, bird, and ancestral
groups of mammals (Supplementary Table S3 online), we revisited the phylogenetic relationships of tetrapod antibody classes
to see if there was now convergence of expression and functional
data suggesting IgX as the mucosal isotype. Entire immunoglobulin heavy chain C region sequences from diverse vertebrates were
used to determine the relationship between amphibian IgX and
mammalian IgA (Figure 5). Sequences were aligned and manually adjusted to maintain domain alignment between isotypes
having either three or four constant domains (Supplementary
Figure S3 online). The resulting trees showed that IgX did not
cluster closest to IgM. Unlike any past phylogenetic analyses, these
data show that IgX and IgA share a common ancestor earlier than
either IgX or IgA does with IgM with high statistical support (91%
MucosalImmunology | VOLUME 6 NUMBER 2 | MARCH 2013
Thymectomy does not impede mucosal antibody production
Normal and thymectomized frogs distinct from those assayed
for gut flora were immunized with dinitrophenol-keyhole limpet
hemocyanin (DNP-KLH) either intracoelomically (IC) or orally
(Figure 6). B cells were harvested and cultured from the spleen
and gut of these animals, and enzyme-linked immunosorbent
assays (ELISAs) were performed for both total IgX and antigen-specific IgX on the supernatant. Oral immunization elicited
significantly (P = 0.025) more total IgX from intestinal B cells
than IC delivery, but no significant difference was seen from the
spleen cells or between B cells from normal and thymectomized
animals. When DNP-KLH-specific IgX was assayed, the only
significant (P = 0.013) difference seen was an increase in specific
IgX from orally immunized splenocytes from thymectomized
frogs vs. orally immunized spleen cells from normal frogs. Thus
larval thymectomy does not appear to retard the frogs’ ability to
make total IgX or IgX specific to this hapten-carrier conjugate.
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51
100
100 M pheasant
M turkey
M quail
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M chicken
85
M duck
M slider
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M turtle
M anole
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M gecko
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M frog
M newt
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M axolotl
M echidna
100
M platypus
M opossum
100
M mouse
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X axolotl
100
Amphibian IgX
X frog
A pheasant
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A turkey
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A duck
IgA bird and
mammal
A echidna
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A opossum
A mouse
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Y pheasant
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100
Y chicken
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IgY reptiles
and birds
Y duck
Y turtle
80
A gecko
Y anole
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70
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E platypus
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100
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mammals
IgE
mammals
E opossum
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100
E brushtail
Y/O platypus
G echidna
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G2 platypus
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IgG mammals
G opossum
G mouse
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100
G human
Y frog
IgY amphibian
Y newt
49
100
Y axolotl
F frog
T zebrafish
84
100
T trout
0.2
Figure 5 Amphibian IgX is orthologous to IgA of birds and mammals. Neighbor-joining phylogenetic tree of the constant regions of diverse
tetrapod immunoglobulin heavy chains, with the fish mucosal isotype IgZ/T, included as an outgroup. Numbers at nodes show bootstrap support
for each bifurcation after 1,000 replications. The alignment used to build this is available as Supplementary Figure S2 online.
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Gut total IgX
0.2
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Figure 6 Thymectomy does not retard induction of mucosal IgX response. Enzyme-linked immunosorbent assay for the IgX isotype on supernatant
of lymphocytes cultured from the spleen or intestine of frogs immunized to dinitrophenol-keyhole limpet hemocyanin. Units of the Y-axes are
absorbance at 450 nm. (a) Oral (p.o.) gives a significantly greater total IgX response in the gut than intracoelomic (IC) immunization (P = 0.025,
marked by *), but no significant difference was seen with thymectomized frogs. No significance was seen monitoring neither antigen-specific IgX in
the gut (b) nor total IgX in the spleen (c). (d) Antigen-specific IgX actually increased from spleen cells with thymectomy (P = 0.013, marked by *).
DISCUSSION
Frog gut flora
Molecular sequencing techniques have surpassed culture methods of investigating gastrointestinal microbiota owing to their
increased sensitivity (and the majority of unculturable genera
present there).21 The amplification and subsequent sequencing of the 16S rRNA gene allows the identification of bacterial
groups present in the gastrointestinal tract of humans and other
animal species.22–24
High-throughput sequencing techniques had not been applied
to the gut microbiota in any amphibian model, and X. laevis’s use
in developmental, cell- and immunobiology made it an obvious
first candidate.25 High-throughput 16S rRNA sequencing has
been used to analyze the antifungal cutaneous bacterial populations in a salamander.26 The most comprehensive culture-based
studies of amphibian gut flora have been performed in the leopard frog (Rana pipiens).27 Similar to the present work in Xenopus,
Rana was found to have many Clostridiaceae, Eubacteriaceae,
and Bacteroidaceae, and hibernating frogs at lower temperatures
had a significant shift of flora to dominant Pseudomonodaceae.28
Using the pyrosequencing-based approach described here, we
were able to identify > 60 families of bacteria in the Xenopus
gut that to our knowledge have not been described previously
in amphibians. These include the known decomposers of plant
polymers Marinilabiaceae,29 the potential pathogens in the guts
of humans and fish Porphyromonadaceae,30 insect endosymbiont Sphingobacteriaceae,31 and the known fermenters of fish
microbiota Verrucomicrobiaceae.32
The frogs in our gut flora analysis all spent their lives in the
same aquatic animal room descended from the same outbred
MucosalImmunology | VOLUME 6 NUMBER 2 | MARCH 2013
founders just one generation before. They all received the same
prepared diet. Although not sterilized, this homogenous, consistent feed is certainly in stark contrast to the varied, inconsistent, and microbe-rich diet wild Xenopus would consume
in Africa. Yet, we saw great individual variance in their gastrointestinal flora, as has been described in humans33 and dogs.34
These communities are undoubtedly temporally dynamic as well
within the individual animals. This work in X. laevis provides a
reference for anatomically discrete gut microbial communities
in an omnivorous amphibian. This is the class of vertebrates that
gave rise to the amniotic reptiles, birds, and mammals, and first
employed immunoglobulin heavy chain isotype switching to a
mucosal isotype.
T-cell influence on mucosal immunity
Formative studies in nude (athymic) mice found that the poorly
developed Peyer’s patches lacking substantial germinal centers and low IgA levels of this rodent model could be largely
restored by thymic grafts and the resulting T-cell population.35
Yet, loss of T-cell function was not found to dramatically alter
the cultivable gastrointestinal microbiota in these mice.36 As
our understanding of T-cell help, class switch recombination
and mucosal immunity have improved in the decades since this
work; much energy has focused on the relationship between the
adaptive immune system and the gut flora. This has extended
to hypotheses of the two coevolving and even rationale for the
original genesis of the adaptive system.37,38
We turned to the major biological model “between” zebrafish
and mammals to ask questions about the influence of T cells
in the mucosal immune compartment. This choice affords a
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comparative view of what the first immune system with both
major histocompatibility complex-restricted T cells and a
humoral response capable of class switch to a mucosal isotype was like in the ancestral tetrapod 300 million years ago.
Thymectomy in this animal is an established model system for
removal of the T-cell compartment,14,39–42 but this study is the
first to rigorously test the adults for T-cell receptor expression
by PCR. We assayed TCR, , and , as TCR has been shown
to be expressed early in the X. laevis thymus.43 Initially, we were
dismayed by some constant region (not necessarily indicative
of functional rearrangement) TCR messenger RNA expression
from peripheral blood of adults in which we visually scored the
thymectomies to be perfectly clean. But perhaps this is to be
expected, as TCR constant region message has been shown
from bone marrow-derived lymphocytes44 and even functional
TCR and TCR transcripts have been found in nude mice.45
Despite these observed low levels of constant domain nucleic
acid expression, we are confident that the scrupulous culling of
tadpoles with incomplete surgeries and molecular diagnostics
of mature animals yielded frogs with no functional T-cell
compartment (and as well). Monoclonal antibodies have confirmed this absence at the cellular level of receptor expression
in this model.46
The possibility of extrathymic T-cell development was
explored in Xenopus by including PCR controls of gut tissue
at the time of thymectomy in addition to the adult gut from
normal and thymectomized frogs (Figure 1). Extrathymic
T-cell development has been described in the mouse gut47 and
may even be stimulated by thymic ablation.48 Highly organized
mucosal lymphoid structures such as Peyer’s patches do not
exist in poikilothermic vertebrates such as Xenopus,49 yet a suggestion of gut T lymphopoiesis has been described from bony
fish.50 Tadpoles showed no TCR expression in the gut at the
age of thymectomy, nor was TCR expression seen in the adult
gut of larval thymectomized frogs. While extrathymic routes
of T-cell development are possible in the frog, our data suggest
that these are at best minor relative to thymic for gut seeding,
and that thymectomy does not force extrathymic developmental
programs.
Some “natural” gut IgA in mouse has been thought to be
from T-independent B-1 cells.51 The specificity of gut IgA was
later shown to be less “natural” and actually driven by specific
antigens of the gut microbial symbionts and food.9 Moreover,
use of TCR/ double knockout transgenic mice ensured that
T cells from thymus or elsewhere were not responsible for this
phenomenon, nor was any “bystander” contribution of their
lymphokines.52 They further showed that this response was
at least in part due to B1 peritoneal cells in mice.9 These findings seem consistent with the lack of effect that thymectomy
has on mucosal IgX in the present study. Yet in mammals,
there is plenty of evidence for T-dependence in gut IgA too.
Most human IgA-switched plasma cells in the lamina propria show evidence of somatic hypermutation, presumably
from a germinal center event with T-cell help.53,54 Moreover,
most (~80%) plasma cells in the gut were found to be antigen specific and not polyreactive. 55 AID transgenic mice
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defective of somatic hypermutation but still competent to
make IgA exhibited greater colony counts of small intestinal flora, germinal center hyperplasia, and susceptibility to
Yersinia enterocolitica.56 This suggests T-dependent, germinal
center processes do shape the gut flora through the specific
IgA generated against it in mice. Deep sequencing of IgA rearrangements in CD3 − / − mice showed evidence for T-dependent somatic hypermutation in aged mice, thus the relatively
young age of the frogs in this study could be a factor.8 We did
not find evidence corroborating such T dependences in the
amphibian, but recognize that this is one relatively small study
in a captive population.
Evolution of mucosal antibody isotypes and IgA
Recent data now allow more rigorous studies of the natural history of tetrapod immunoglobulin genes. Sequence similarities
and predicted structural resemblance to IgM originally suggested
that IgX might be the functional analog, but not the ortholog
of IgA.16,17 The IgA of Aves appears to be a mucosal functional
analog of mammalian IgA57 and there is high sequence identity
that suggests orthology.58 However, there are four C domains
in avian IgA suggesting a deletion occurred to yield the mammalian IgA of three.59 The incomplete evolutionary loss of
the C2 domain present in IgX and avian IgA could have given
rise to the hinge region in mammalian IgA before the divergence of monotremes and the therian marsupial and placental
mammals.60
Although IgX has been hypothesized to be an ortholog of
IgA,15,61 the phylogenetic analysis described here is the first
to lend experimental support to the notion. The availability
of more diverse tetrapod immunoglobulin sequences allowed
us to make this analysis. The CH1- and CH2-encoding exons
of the IgX gene may have been derived from the IgY-encoding locus and the CH3 and CH4 from the IgM-encoding
gene. 62,63 These relationships will need to be retested as
more amphibian, reptile, bird, and non-placental mammal
genomes are sequenced. However, this scenario is consistent with the known synteny of the C-region-encoding genes
in the immunoglobulin heavy chain loci of known tetrapod
genomes (Supplementary Figure 4 online), where IgX/A is
in between the genes encoding the IgM/D- and IgY-encoding
loci that birthed it. The locus in mammals has undergone
duplicative expansions giving rise to subfunctionalization of
IgG and IgE from IgY and a proliferation of sub-isotypes.64
Thus, the resulting IgY/IgM chimera IgX gave rise to (or perhaps should be synonymous with) IgA in endothermic vertebrates. This appears to be the second time vertebrate evolution
has produced a dedicated mucosal immunoglobulin isotype
(Figure 7), the first being IgZ/T that is unique to some teleost
fish.65–67 IgX/A is the first mucosal isotype whose expression
is controlled via AID-mediated class switch recombination,
as IgZ/T is produced via deletional RAG-mediated V(D)J
recombination similar to the rearrangement at the / T-cell
receptor locus.68,69
In the frog, we find evidence for an ancient, T-independent,
humoral mucosal response. We defined the gut flora of the
VOLUME 6 NUMBER 2 | MARCH 2013 | www.nature.com/mi
ARTICLES
X
A
Jawless
fish
Cartilaginous
fish
Bony
fish
Amphibians Reptiles
Birds
G
Mammals
M
T
Y
MYA
E
D
160-
Previous consensus
A
300-
X
G
350380450520-
T
M
D
Y
E
Revised model
Figure 7 Model of immunoglobulin natural history with mucosal IgX/A emerging in early tetrapods. (a) Simplified phylogeny of vertebrates showing
approximate emergence times of heavy chain isotypes. (b) This analysis suggests that the isotype previously described as IgX in amphibians may be
orthologous to IgA of warm-blooded vertebrates (model adapted from M Flajnik’s chapter of Fundamental Immunology, W. Paul editor). In addition to
the isotypes extant in man, the immunoglobulin light chain-less IgNAR of cartilaginous fish and the mucosal IgZ/T of bony fish are also shown.
model amphibian X. laevis but found the bacterial communities of the stomach, small and large intestine to be unaffected by
thymectomy. More representative phylogenetic analysis of the
relationship between amphibian IgX and IgA of amniotic tetrapods shows their orthology, explaining the origin of human’s
most abundant immunoglobulin. Therefore, we conclude that
the T-cell-independent IgA pathway is likely an ancient mechanism to manage the microbial symbionts of the gut and other
mucosal surfaces. More comparative studies must resolve the
(convergent?) functional relationship between the two vertebrate mucosal isotypes IgZ/T and IgX/A.
METHODS
Animals. X. laevis was used as a model for the tetrapod vertebrate
immune system. Outbred frogs were initially purchased from Xenopus
Express (Brooksville, FL). Subsequent generations were bred in-house
using human chorionic gonadotropin hormone to prime for egg and
sperm maturation (Sigma-Aldrich, St Louis, MO). Frogs were maintained at the Texas A&M Comparative Medicine Program facility.
They were housed in two separate but similar recirculating rack systems (Techniplast, Buguggiate, Italy) on a 12-h light cycle. Frogs were
moved from an antigen-free system to a “DNP-KLH exposed” system
upon first immunization. Adults were fed a sinking pellet and tadpoles a
powder diet (Xenopus Express). All husbandry, surgery, and immunization protocols were approved by the Texas A&M Institutional Animal
Care and Use Committee (AUP 2008-33). Post-metamorphosis frogs
were microchipped (Avid, Norco, CA) and assigned to gut microbiota
harvest or immunization protocols.
Thymectomy. Frogs were thymectomized 9 days post fertilization
through microscopic cauterization, adapting the protocol devised by
Horton.19 The surgery was performed under a dissecting microscope
using a micro-cautery apparatus originally designed for insect stylectomy
(http://aphidzapper.com), delivering a VHF pulse of 10-ms duration
and power of 10 W via an abraded tungsten wire to the target tissue. Tadpoles were anesthetized using a 300 mg l − 1 MS222 (tricaine
methanesulfonate, Argent, Redmond, WA) bath for 2–10 min, before
placing on a wet cool cheesecloth over a grounded metal plate stage.
The thymus was then burned with one pulse on each side. The thymus
is located bilaterally caudal and medial to the eyes and lateral to the dark
MucosalImmunology | VOLUME 6 NUMBER 2 | MARCH 2013
central nervous system (Figure 1a). The tadpole was transferred to an ice
bath immediately after thymic ablation to cool the animal for 3 s before
being allowed to recover in an aerated tank with 80 mg l − 1 carbenicillin
(and no MS-222) to prevent superficial infections. After 24 h, tadpoles
were transferred back to the primary recirculating Xenoplus systems
(with no antibiotic). Three days after surgery, tadpoles were monitored
for any regrowth of the thymus by visual inspection for the melanized
organ under the microscope (~20% of frogs that recover from surgery).
The monitoring continued every 4–6 days for 1 month post surgery.
Tadpoles with thymic regrowth owing to incomplete thymectomy were
euthanized.
PCR validation of thymectomy. Frogs thymectomized as tadpoles
were checked for the presence of TCR, , and messenger RNA
using PCR at least 6 months post surgery (they undergo metamorphosis in the second month, Supplementary Table S1 online). The more
exposed tarsal veins associated with digits two and three were bled for
100–500 l with 1-ml syringes and 28 gauge needles. Additional checks
for TCR expression were performed on the gut of 9-day tadpoles, the
gut of thymectomized adult, and the gut of normal adult. PCR was performed on complementary DNA prepared from RNA prepared from
whole peripheral blood, owing to the small volumes of blood collected
using RNAeasy preparations (Qiagen, Valencia, CA) with on-column
genomic DNA digestion. First-strand complementary DNA was synthesized using random hexamer priming with Superscript III (Invitrogen,
Carlsbad, CA) according to the manufacturer’s protocol. PCR amplification was performed using oligonucleotide primers designed for constant
domain genes of TCR, , , and 2M (Supplementary Table S2 online).
Gotaq polymerase (Promega, Madison, WI) and 50 ng template were
used during these PCR for 35 cycles for blood and 2 g template for 39
cycles for intestine. Primer sets for TCR and 2m annealed at 52 °C and
and at 58 °C.
DNA preparation and 16S rRNA gene sequencing. Gut contents were
sampled from normal and thymectomized frogs at three anatomical sites:
stomach, small intestine, and large intestine (as shown in Supplementary
Figure S1 online). Bolus and chyme were scraped from longitudinally
opened organs and collected as ~300-l samples for DNA isolation via
phenol–chloroform–isoamyl alcohol extraction.34 Bacterial tag-encoded
FLX-titanium amplicon pyrosequencing (bTEFAP) was performed similarly as described previously at the Research and Testing Laboratory,
Lubbock, TX,70 but based on the V4-V6 region (E. coli position
365
ARTICLES
530–1,100) of the 16S rRNA gene, with primers forward 530F and reverse
1100R (Supplementary Table S2 online). Briefly, the DNA concentration
was determined using a Nanodrop spectrophotometer (Nyxor Biotech,
Paris, France). A 100-ng (1 l) aliquot of each DNA sample was used for
a 50-l PCR reaction. HotStarTaq Plus Master Mix Kit (Qiagen) was
used for PCR under the following conditions: 94 °C for 3 min followed by
32 cycles of 94 °C for 30 s; 60 °C for 40 s; and 72 °C for 1 min; and a final
elongation step at 72 °C for 5 min. A secondary PCR was performed for
FLX (Roche, Nutley, NJ) amplicon sequencing under the same conditions
by using designed special fusion primers with different tag sequences as
LinkerA-Tags-530F and LinkerB-1100R. The use of a secondary PCR
prevents amplification of any potential bias that might be caused by inclusion of tag and linkers during initial template amplification reactions.
After secondary PCR, all amplicon products from the different samples
were mixed in equal volumes, and purified using Agencourt Ampure
beads (Agencourt, Danvers, MA).
Gut flora analysis. Raw sequence data were screened, trimmed, filtered, denoised, and chimera depleted with default settings using the
QIIME pipeline version 1.4.0 (http://qiime.sourceforge.net)71 and with
USEARCH using the operational taxonomic unit pipeline (www.drive5.
com). Operational taxonomic units were defined as sequences with at
least 97% similarity using QIIME. For classification of sequences on a
genus level, the naïve Bayesian classifier within the Ribosomal Database
Project (RDP, v10.28, East Lansing, MI) was used. The compiled data were
used to determine the relative percentages of bacteria for each individual
sample, 6 frogs by 3 samples each for a total of 18. To account for unequal
sequencing depth across samples, subsequent analysis was performed on
a randomly selected subset of 1,800 sequences per sample. This number
was chosen to avoid exclusion of samples with lower number of sequence
reads from further analysis. Alpha diversity (i.e., rarefaction) and beta
diversity measures were calculated and plotted using QIIME. Differences
in microbial communities between disease groups were investigated using
the phylogeny-based unweighted Unifrac distance metric. This analysis
measures the phylogenetic distance among bacterial communities in a
phylogenetic tree, and thereby provides a measure of similarity among
microbial communities present in different biological samples.
IgX phylogenetics. Amino-acid sequences of tetrapod immunoglobu-
lin heavy chain constant regions were compiled and aligned in BioEdit
with ClustalW employing gap opening penalties of 10 and gap extension penalties of 0.1 for pairwise alignments and then 0.2 for multiple
alignments with the protein-weighting matrix of Gonnett or Blossum.72,73
These alignments were then heavily modified by hand. MEGA was
used to infer the phylogenetic relationships of the immunoglobulin
heavy chain constant genes. Evolutionary distances were computed
using the Dayhoff matrix74 and 509 column positions in the 56
selected sequences. Several tree-building algorithms were employed,
including a consensus neighbor-joining tree made from 1,000 bootstrap
replicates.
Immunizations. For immunization, two routes of administration were
used. Four frogs received IC (frogs have no peritoneal cavity) and four
oral (p.o.) immunization. We used an oral gavage needle to deliver conjugated DNP-KLH (Calbiochem, San Diego, CA) for mucosal immunization as previously described for X. laevis.15 There were two normal and
two thymectomized frogs in each group for a total of eight frogs. Frogs
orally immunized received 2.5 mg DNP/KLH with 10 g cholera toxin as
adjuvant three times, each at weekly intervals. Animals in the IC group
received 200 g of antigen with equal volume (200 l) of Freund’s complete adjuvant once and Freund’s incomplete adjuvant twice, at weekly
intervals.
mesh inundated with amphibian-adjusted phosphate-buffered saline
(PBS). The intestine was excised below the stomach and above the
rectum. We removed mucous, chime, and fecal matter by physically
scraping and flushing the organ with PBS. The intestine was cut into
smaller pieces (~0.5 cm2) and placed in PBS. We then added 2% collagenase (Calbiochem) in amphibian PBS to aid in isolation of the intestinal
lymphocytes. We allowed the intestine to digest for 120 min at room temperature. It was vortexed initially and once every 30 min. The intestine
was strained through a 100 m nylon cell strainer (BD Falcon, San Jose,
CA) at the end of the 120 min. Lymphocytes were isolated from the
washed supernatant of the intestine and from the cells isolated from the
spleen with Lymphocyte Separation Medium (Mediatech, Manassas, VA).
All intestine and spleen cells isolated were counted (Supplementary
Figure S2 online) and cultured in 24-well plates containing 250 l of
L-15 media with 10% fetal calf serum at a density of 2.3×106 cells per
ml for the spleen cells and 1.0×106 cells per ml for the intestinal cells.
Cultures were incubated at humidified 28 °C and 5% CO2. On the third
day, supernatant was collected and new media was added to maintain a
volume of 250 l per well. On the sixth day, supernatant was once again
collected and pooled with the day 3 collections.
Enzyme-linked immunosorbent assay. We used ELISAs to determine
the total (non-antigen specific) and antigen-specific levels of amphibian
mucosal antibody isotype IgX produced in response to DNP-KLH. Serial
dilutions from 1/10 to 1/1011 were made from the supernatants of the
spleen and intestinal cultures. For the total immunoglobulin ELISAs,
we added 100 l of each dilution to the well of a plate and incubated for
1 h at 37 °C. The plate was then washed 2 times with 200 l PBS, and
200 l of 2% casein in PBS was added as blocking solution. The plate was
allowed to sit overnight at 4 °C, and was then washed 3 times with 200 l
PBS. We added mouse anti-Xenopus IgX monoclonal 4110B3 (kind gifts
of Martin Flajnik, University of Maryland at Baltimore and Louis Du
Pasquier, University of Basel) to each well to obtain a volume of 100 l.75
The plate was allowed to incubate at room temperature for 1 h and then
was washed 4 times with 200 l PBS with 0.05% Tween-20 (PBS-T, Sigma,
St Louis, MO). The wells received 100 l of anti-mouse IgG peroxidaseconjugated secondary antibody (Sigma) and were incubated for 1 h at
room temperature. We then washed the plate 4 times with 200 l PBS-T
and 1 time with PBS. A 3,3⬘,5,5⬘-tetramethylbenzidine substrate solution
was then added to each well and the reaction was allowed to take place for
3 min before being stopped with 2 M H2SO4. Plates were read at an optical
density of 450 nm in a BioRad iMark Microplate Reader (Hercules, CA).
We used the 104 dilution of supernatant for all trials. The antigen-specific
ELISAs used a similar protocol, except 100 l of 10 g ml − 1 DNP-KLH
was added to each well for initial coating. After blocking overnight, serial
dilutions of the sample supernatant were added and allowed to incubate
for 2 h at 37 °C. The remaining protocol was the same as that for total
IgX. We assayed wells in triplicate, showed the s.e.m. and employed a
Student’s t-test.
SUPPLEMENTARY MATERIAL is linked to the online version of the
paper at http://www.nature.com/mi
ACKNOWLEDGMENTS
This work was supported by the NIH through a K22 award to MFC
(AI56963) and a graduate student grant from the Texas A&M College of
Veterinary Medicine to SM. AG was supported by the National Center for
Research Resources and the Office of Research Infrastructure Programs
(ORIP) of the National Institutes of Health through Grant Number 5 T35
RR019530-07. We appreciate the expert training in tadpole thymectomy
provided by Martin Flajnik.
Lymphocyte isolation and culture. Three weeks after the last boost, we
DISCLOSURE
The authors declare no conflict of interest.
euthanized frogs with an MS-222 overdose and decapitation. Spleens
were removed and cells dissociated by scraping the organs over a wire
© 2013 Society for Mucosal Immunology
366
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ARTICLES
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